ABSTRACT Currently, the only known strategy to cure HIV/AIDS is allogeneic hematopoietic stem cell transplantation (allo-HSCT) from a donor with the HIV-resistant homozygous CCR5?32 mutation. And while this procedure may have cured 3 individuals as of March 2019, it is only appropriate in cases were an underlying hematologic malignancy warrants the significant associated toxicities. The combined effects of conditioning regimens and graft versus host from the donor immune system are believed to deplete the reservoir of HIV-infected cells that persists even under antiretroviral therapy, while the new donor derived immune cells resist HIV infection. For a successful cure in HIV-infected individuals without malignancies it will be necessary to develop a less toxic autologous HSCT approach that still incorporates the major components of the CCR5?32 allo-HSCT cures. In this U19 we aim to develop an autologous HSCT approach that takes advantage of advances made by the program investigators in hematopoietic stem and progenitor cell (HSPC) engineering, nongenotoxic conditioning regimens and enhanced T cell development post-transplantation. Our approach will exploit powerful genome editing technologies to make individual cells resistant to HIV, activate intrinsic cellular defense mechanisms, secrete broadly acting entry inhibitors and neutralizing antibodies, and engineer T cell responses to recognize infected cells. Prior work by Drs. Cannon (Project 3) and Kiem (Project 4) has already demonstrated that genetically engineered CCR5 disruption provides HIV control in animal models, and this will now be combined with additional strategies to further block infection using entry inhibitors, and to enhance CAR T cell immune responses. These will be combined with conditioning regimens developed by our biotech partner, Magenta Therapeutics (Project 2), based on nongenotoxic antibody-drug conjugates (ADCs). By replacing current high-dose or myeloablative conditioning regimens with nongenotoxic ADCs, we expect to reduce toxicity and better preserve existing anti-HIV immunity, while the ADC conditioning may also impact the latent reservoir. Finally, we will improve the development of T cell progeny from engineered autologous HSPC by providing a cryogel biomimetic of the bone marrow environment (Project 1). This is expected to be an important adjunct to HSCT, that may help overcome the defects in the T cell compartment in HIV-infected individuals and enhance repopulation by HIV-resistant progeny. Together, these strategies will be tested in an escalating series of animal models that the investigators in this U19 have developed, based on humanized mice and nonhuman primates (NHPs) and supported by an NHP core. Finally, in a direct test of how this therapy could impact HIV-infected individuals on ART, we will perform autologous HSCT with gene-engineered HSPC in SHIV-infected ART- suppressed NHPs.